1. Field of the Invention
This invention relates to the human pancreatic cholesterol esterase gene. In particular, the invention relates to the identification of restriction fragment length polymorphisms (RFLP) of the human pancreatic cholesterol esterase gene. Specifically, the invention relates to the use of RFLP analysis for identifying individuals with a particular genetic variant of the human pancreatic cholesterol esterase gene. The invention also relates to development of methods for identifying individuals for appropriate treatment with therapeutic drugs for the prevention or alleviation of disease states in a human related to cholesterol metabolism.
Cholesterol metabolism is of critical interest to those involved in protecting human health. Atherosclerosis is the leading cause of death in the United States and reduction of serum cholesterol levels has recently been embraced as a national health priority. See NIH Consensus Panel Report, J.A.M.A. 253: 2094 (1985). NIH recommendations include measurement of serum cholesterol in all adults, with efforts to reduce cholesterol in those individuals with levels above 200 mg %. In this regard front line therapy is a reduction in the amount of cholesterol and triglycerides ingested, followed by the use of agents that interfere with absorption of ingested lipids. See Consensus Full Report, Arch. Inst. Med. 148: 36 (1988).
Since free cholesterol comprises about 90% of dietary cholesterol, it is not obvious that knowing either the phenotype or the genotype of the pancreatic cholesterol esterase gene would be useful. In fact, it had been thought prior to this time that cholesterol esterase was not important for cholesterol absorbsion [Huang and Hiu, J. Lipid Res. 31: 2029 (1991)]. Unexpectedly, pancreatic cholesterol esterase plays a pivotal role in the absorption of cholesterol and fatty acids (U.S. Pat. No. 5,017,565, issued May 21, 1991. Alterations in the genotype or phenotype of this enzyme may be a factor responsible for differences among individuals in susceptibility for developing cardiovascular disease and/or lipid abnormalities.
One way of investigating such genotypic alterations is the use of restriction fragment length polymorphism (RFLP) analysis. Using this technique, DNA polymorphisms can be detected as differences in the length of DNA fragments after digestion with DNA sequence-specific restriction endonucleases. Restriction fragments can then be separated by agarose gel electrophoresis, according to their molecular size, to reveal a pattern of RFLP-related bands. Differences in the length of a particular fragment may result from individual or multiple base substitutions, insertions or deletions. These genotypic changes can be recognized by the altered mobility of restriction fragments on agarose gel electrophoresis. Specific DNA sequences can then be detected by hybridization with a complimentary radioactive probe [see, for example, Botstein et al. Am. J. Hum. Genet. 32: 314-331 (1980)].
RFLP analysis has been used in studying a number of genes believed to be involved in either the absorbsion, transport or metabolism of cholesterol in vivo. These include the low density lipoprotein (LDL) cholesterol receptor gene relating to the diagnosis of familial hypercholesterolemia (FH) [see, for example, Lehrman et al., Proc. Natl. Acad. Sci. USA 83: 3679-3683 (1986); Hobbs et al., J. Clin. Invest. 81: 909-917 (1988); Daga et al., Hum. Genet. 84: 412-416] and the genes of apolipoproteins A-I, A-IV, and C-III [see, Karathanasis et al., Nature 304: 371-373 (1983); Karathanasis et al., Nature 305: 823-825 (1983); Shaw et al., Hum. Genet. 74: 267-269 (1986); Johansen et al., Clin. Genet. 37: 194-197 (1990); Funke et al., J. Clin. Invest. 87: 371-376 (1991)], all of which are associated with premature coronary heart disease [Antonakis, N.E.J.M. 320: 153-163 (1989)].
The present invention provides methods and reagents for detecting RFLPs in the human pancreatic cholesterol esterase gene. In addition, the invention provides methods and reagents for identifying RFLPs in this gene. Specifically, the invention relates to a RFLP in the gene that is related to a particular phenotype associated with the cholesterol esterase gene. This invention also provides methods and reagents for screening a human population for a particular RFLP and for identifying a target patient population for treatment of cholesterol esterase-related disease.
2. Information Disclosure Statement
Borja et al., Proc. J. Exp. Biol. and Med. 116: 496 (1964) teach that cholesterol esterase is secreted by the pancreas, and that its catalysis of cholesterol ester hydrolysis to produce free cholesterol and free fatty acids is essential for the absorption of cholesterol derived from cholesterol esters.
Botstein et al., Am. J. Hum. Genet. 32: 314-331 (1980) teach the use of restriction fragment length polymorphisms (RFLPS) for genetic mapping.
Norum et al., Physiol. Rev. 63: 1343-1419 (1983) review the biochemistry of cholesterol absorbsion and metabolism, including the role of pancreatic cholesterol esterase.
Karathanasis et al., Nature 304: 371-373 (1983) disclose RFLPs in the human apolipoprotein A-I and C-III genes.
Karathanasis et al., Nature 305: 823-825 (1983) disclose an RFLP in the human apolipoprotein A-I gene.
Lehrman et al., Proc. Natl. Acad. Sci. USA 83: 3679-3683 (1986) teach the association between an RFLP in the low density lipoprotein gene (LDL) and familial hypercholesterolemia (FH).
Bosner et al., Proc. Natl. Acad. Sci. USA 85: 7438-7442 (1988) teach that cholesterol esterase performs its function while anchored to the intestinal membrane via a receptor-like interaction with brush border membrane associated heparin.
Hobbs et al., J clin. Invest. 81: 909-917 (1988) disclose RFLPs associated with FH.
Cooper & Clayton, Hum. Genet. 78: 299-312 (1988) teach the use of RFLP analysis for the diagnosis of genetic disease and review the association between genetic polymorphism in apolipoprotein genes and atherosclerosis.
Ordovas & Schaefer, Ann. Biol. Clin. (Paris) 46: 24-29 (1988) teach the relationship between an RFLP in a PstI site in the human apolipoprotein A-I gene and coronary artery disease.
Kyger et al., Biochem. Biophys. Res. Comm. 164: 1302-1309 (1989) teach the nucleic acid sequence of a cDNA clone of bovine pancreatic cholesterol esterase.
Kissel et al., Biochim. Biophys. Acta 1006: 227-236 (1989) teach the nucleic acid sequence of a cDNA clone of mRNA encoding pancreatic cholesterol esterase of the rat.
Nilsson et al., Eur. J. Biochem. 192: 323-326 (1990) teach the nucleic acid sequence of a partial cDNA clone of human pancreatic cholesterol esterase.
Daga et al., Hum. Genet. 84: 412-416 (1990) disclose a RFLP in the LDL receptor gene associated with FH.
Johansen et al., Clin. Genet. 37: 194-197 (1990) disclose RFLPs in the apolipoprotein A-I/C-III gene cluster.
Berg, Acta Genet. Med. Gemellol. (Roma) 39: 15-24 (1990) review the association between RFLPs in human apolipoprotein genes and coronary heart disease.
Taylor et al., Genomics 10: 425-431 (1991) disclose the localization of the human cholesterol esterase gene to the terminal region of the long arm of chromosome 9.
Funke et al., J. Clin. Invest. 87: 371-376 (1991) disclose an RFLP in the human apolipoprotein A-I gene related to disease.